The present invention relates generally to isotopically enriched nucleic acids, and methods for the production and purification thereof. More particularly, the present invention relates to isotopically enriched nucleic acids, and methods for the production and purification thereof for use in analysis with mass spectrometry.
Mass spectrometry is a well-known and widely used tool for analyzing and identifying chemical compositions. Electrospray ionization (ESI) mass spectrometry and matrix-assisted laser desorption ionization (MALDI) mass spectrometry have been used to analyze, with varying degrees of sensitivity, a wide variety of materials including large chemical entities, e.g. biomolecules. Sufficient spectrometric sensitivity and resolution of many large molecules, especially nucleic acids and proteins, has proven difficult for a variety of reasons. One such difficulty arises from the existence of different naturally occurring isotopes for chemical elements contained therein, especially for carbon (C), nitrogen (N), and oxygen (O). For large biomolecules, the statistical distribution of naturally-occurring isotopes results in a mass spectrum with broad peak widths, in which small differences in mass are difficult to resolve. This problem is compounded with increasing size of the molecule.
Attempts to prepare biomolecules compatible with mass spectrometry analysis continue to be sought through on-going research and development efforts.
The present invention is directed to nucleic acids isotopically enriched in an isotope of oxygen. Another aspect of the invention relates to methods for the production and purification of such isotopically enriched nucleic acids. Another aspect of the invention relates to methods for the use of said nucleic acids in mass spectrometric analysis. In still another aspect of the invention, the use of isotopically enriched nucleic acids to create second generation products, such as their use in polymerase chain reactions (PCRs) is described.
One aspect of the invention relates to nucleotides and oligonucleotides, wherein said nucleotides and oligonucleotides are isotopically enriched in one of the isotopes of oxygen. Another aspect of the invention relates to nucleotides and oligonucleotides, wherein said nucleotides and oligonucleotides are isotopically enriched in one or more of the constituent chemical elemental isotopes selected from the group consisting of oxygen (O); oxygen and carbon (C); and oxygen, carbon, and nitrogen (N). Another aspect of the invention relates to nucleotides and oligonucleotides, wherein said nucleotides and oligonucleotides are isotopically enriched in one or more of the constituent chemical elemental isotopes selected from the group consisting of 16O and 12C; and 12C, 14N and 16O. Nucleotides and oligonucleotides of the invention which are enriched in oxygen may also be enriched in other isotopes of chemical elements such as 12C and 15N. Another aspect of the invention is a method for the preparation of a nucleotide that is isotopically enriched in one or more of the constituent chemical elemental isotopes selected from the group consisting of 16 O; 16O and 12C; and 12C, 16O and 14N, the method comprising the steps of: (a) producing DNA or RNA in an organism grown in a growth medium in which the nutrients, reagents and solvents are isotopically enriched in one or more of the constituent chemical elemental isotopes selected from the group consisting of 16O; 16O and 12C; and 12C, 14N and 16O; (b) isolating the isotopically enriched DNA or RNA; (c) hydrolyzing the isotopically enriched DNA or RNA in 16O enriched water. In one aspect, the nucleotide is enriched in an isotope of oxygen.
Another aspect of the invention is a method for determining the mass of an oligonucleotide that is isotopically enriched in one or more of the constituent chemical elemental isotopes selected from the group consisting of 16O; 16O and 12C; and 12C, 14N and 16 O, the method comprising the steps of: (a) making an oligonucleotide isotopically enriched in one or more of the constituent chemical elemental isotopes selected from the group consisting of 16O; 16O and 12C; and 12C, 14N and 16O; (b) determining the mass of the isotopically enriched DNA or RNA using mass spectrometry. In one aspect, the oligonucleotide is enriched in an isotope of oxygen.
Other features of the present invention will become clearer from the following detailed description of the invention, taken with the accompanying drawings and claims.
The entire disclosures of the publications and references referred to in this specification are incorporated herein by reference in their entirety.